Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl− was the primary charge carrier (termed I Cl,swell). The selectivity sequence of the underlying volume-regulated anion channels (VRACs) for different anions was Br−≈I− > Cl− > F− > methanesulfonate ≫ glutamate, with relative permeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currents as well as single-channel currents showed moderate outward rectification. Unitary VRAC conductance was determined at 9.6 ± 1.8 pS. Conventional Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM) and DIDS (100 μM) inhibited whole cell I Cl,swell in a voltage-dependent manner, with the block decreasing from 39.6 ± 9.7% and 71.0 ± 11.0% at +50 mV to 26.2 ± 7.2% and 14.5 ± 6.6% at −100 mV, respectively. Verapamil (50 μM), a standard Ca2+ antagonist and P-glycoprotein function inhibitor, depressed the current by a maximum of 15%. Protein tyrosine kinase inhibitors downregulated I Cl,swell(genistein with an IC50 of 2.6 μM and lavendustin A by 60 ± 14% at 1 μM). The protein tyrosine phosphatase inhibitor sodium orthovanadate (500 μM) stimulated I Cl,swell by 54 ± 11%. We conclude that VRACs in human prostate cancer epithelial cells are modulated via protein tyrosine phosphorylation.